GNK and sGNK: A Kinase and its Putative Polypeptide Substrate
The isolation of a novel protein kinase, GNK, has been recently disclosed [Sims et al., WO 97/47750]. GNK was originally identified as an IL-1 stimulated kinase and was initially named ITAK (IL-1/TNF-α activated Kinase). However, GNK, which is short for GEF containing NEK-like Kinase, was subsequently renamed for its structural/catalytic components, i.e., it contains both (i) an N-terminal kinase domain most similar to that of protein kinase Nek-2, and (ii) a domain that is homologous to the Guanine nucleotide Exchange Factor (GEF) family of proteins [U.S. Pat. No. 6,080,557, Issued Jun. 27, 2000, WO 97/47750, and WO 00/36097 the contents of which are hereby incorporated by reference in their entireties]. A polypeptide substrate for GNK, which was named sGNK, was found to co-purify with the protein kinase. Both GNK and sGNK appear to play a role in the regulation of vascularization during embryonic development.
GNK has an approximate molecular weight of 110 kDa, and is capable of phosphorylating polypeptide substrates such as sGNK, as well as undergoing autophosphorylation. Phosphorylated-GNK demonstrates a strong tendency to oligomerize. Based on SDS-PAGE and SUPERDEX 200 size exclusion chromatography analyses, phosphorylated-GNK forms trimers and also higher-order complexes.
As indicated above, GNK has both a kinase domain and a GEF-like domain. The kinase domain of GNK is most similar to the NIMA family of kinases, particularly Nek2 (NIMA-related kinase 2). The Nek2 kinase is a dual specificity kinase associated with regulation of the cell cycle. Nek2 associates with the centrosomes of all cells during all stages of the cell cycle and has been shown to be a bona fide component of the core centrosome [Fry et al., EMBO J. 17:470-481 (1998)]. GEF polypeptides are activators of the Ras superfamily of proteins [Overbeck et al., Mol. Repro. and Dev., 42:468, (1995)], which play a role in the regulation of a wide variety of cellular activities, such as cell proliferation and differentiation, cytoskeletal organization, nuclear transport, and the cell cycle. Ras superfamily proteins are GTP binding proteins which are active when bound to GTP, but become inactive when the GTP is hydrolyzed to GDP. GEFs positively regulate Ras activity by promoting the release of bound GDP, thereby facilitating GTP binding and Ras activation.
sGNK is approximately 90 kilodaltons (kDa), appears to have a high degree of coiled-coil structure, and appears to be the human homologue of the Drosophila bicaudal-D protein. sGNK also has a region of similarity with a newly discovered polypeptide, C-Nap1. Mutations in bicaudal-D disrupt the cytoskeleton, interfere with messenger RNA (mRNA) sorting, and disrupt the polarity of the developing Drosophila embryo [Baens and Marynen, Genomics, 45:601-606 (1997)]. C-Nap1 is a novel centrosomal coiled coil protein that appears to be the substrate of Nek2 [Fry et al., J. Cell Biol. 141:1563-1574 (1998)]. C-Nap1, like Nek2, is a core component of the human centrosome, that associates with centrosomes independently of the microtubules [Fry et al., J. Cell Biol. 141:1563-1574 (1998)]. C-Nap1 and Nek2 are known to co-localize in the centrosome and both have been detected in all cell types examined. A recent model suggests that C-Nap1 may function as part of the centrosomal “glue”, by linking the ends of centrioles to each other during interphase. C-Nap1 is believed to be phosphorylated by Nek2 at the onset of mitosis, causing C-Nap1 to depolymerize or break down, which in turn permits the centrosomes to split during mitosis. Since sGNK is phosphorylated by GNK in vitro, the interaction between GNK and its substrate sGNK may resemble that observed with Nek2 and C-Nap1.